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Fission Yeast Schizosaccharomyces Pombe (fission + yeast_schizosaccharomyces_pombe)
Selected AbstractsMolecular interaction of neutral trehalase with other enzymes of trehalose metabolism in the fission yeast Schizosaccharomyces pombeFEBS JOURNAL, Issue 15 2002Teresa Soto Trehalose metabolism is an essential component of the stress response in yeast cells. In this work we show that the products of the principal genes involved in trehalose metabolism in Schizosaccharomyces pombe, tps1+ (coding for trehalose-6- P synthase, Tps1p), ntp1+ (encoding neutral trehalase, Ntp1p) and tpp1+ (that codes for trehalose-6- P phosphatase, Tpp1p), interact in vitro with each other and with themselves to form protein complexes. Disruption of the gene tps1+ blocks the activation of the neutral trehalase induced by heat shock but not by osmotic stress. We propose that this association may reflect the Tps1p-dependent requirement for thermal activation of trehalase. Data reported here indicate that following a heat shock the enzyme activity of trehalase is associated with Ntp1p dimers or trimers but not with either Ntp1p monomers or with complexes involving Tps1p. These results raise the possibility that heat shock and osmotic stress activate trehalase differentially by acting in the first case through an specific mechanism involving Tps1p,Ntp1p complexes. This study provides the first evidence for the participation of the catabolic enzyme trehalase in the structural framework of a regulatory macromolecular complex containing trehalose-6- P synthase in the fission yeast. [source] Characterization of active-site mutants of Schizosaccharomyces pombe phosphoglycerate mutaseFEBS JOURNAL, Issue 24 2000Elucidation of the roles of amino acids involved in substrate binding, catalysis The roles of a number of amino acids present at the active site of the monomeric phosphoglycerate mutase from the fission yeast Schizosaccharomyces pombe have been explored by site-directed mutagenesis. The amino acids examined could be divided broadly into those presumed from previous related structural studies to be important in the catalytic process (R14, S62 and E93) and those thought to be important in substrate binding (R94, R120 and R121). Most of these residues have not previously been studied by site-directed mutagenesis. All the mutants except R14 were expressed in an engineered null strain of Saccharomyces cerevisiae (S150-gpm::HIS) in good yield. The R14Q mutant was expressed in good yield in the transformed AH22 strain of S. cerevisiae. The S62A mutant was markedly unstable, preventing purification. The various mutants were purified to homogeneity and characterized in terms of kinetic parameters, CD and fluorescence spectra, stability towards denaturation by guanidinium chloride, and stability of phosphorylated enzyme intermediate. In addition, the binding of substrate (3-phosphoglycerate) to wild-type, E93D and R120,121Q enzymes was measured by isothermal titration calorimetry. The results provide evidence for the proposed roles of each of these amino acids in the catalytic cycle and in substrate binding, and will support the current investigation of the structure and dynamics of the enzyme using multidimensional NMR techniques. [source] The epigenetic calnexin-independent state is induced in response to environmental changesFEMS YEAST RESEARCH, Issue 8 2009Renée Guérin Abstract Yeasts have evolved numerous responsive pathways to survive in fluctuating and stressful environments. The endoplasmic reticulum (ER) is sensitive to adverse conditions, which are detected by response pathways to ensure correct protein folding. Calnexin is an ER transmembrane chaperone acting in both quality control of folding and response to persistent stress. Calnexin is a key protein required for viability in certain organisms such as mammals and the fission yeast Schizosaccharomyces pombe. Nevertheless, S. pombe calnexin-independent (Cin) cells were obtained after transient expression of a particular calnexin mutant. The Cin state is dominant, is stably propagated by an epigenetic mechanism and segregates in a non-Mendelian fashion to the meiotic progeny. The nucleolar protein Cif1p was identified as an inducer of the Cin state in a previous genetic screen. Here, we report the identification of novel inducers isolated in an overexpression genetic screen: pyruvate kinase (Pyk1p) and phosphoglycerate kinase (Pgk1p). Addition of pyruvate, the end product of pyruvate kinase and glycolysis, also induced calnexin independence in a dose-dependent manner. Remarkably, growth in respiration media or cold temperatures induced the appearance of Cin cells at high frequencies. Taken together, our results indicate that the Cin state can be triggered by extracellular changes, suggesting that this state represents an epigenetic adaptative response to environmental modifications. [source] Pgt1, a glutathione transporter from the fission yeast Schizosaccharomyces pombeFEMS YEAST RESEARCH, Issue 6 2008Anil Thakur Abstract The Schizosaccharomyces pombe ORF, SPAC29B12.10c, a predicted member of the oligopeptide transporter (OPT) family, was identified as a gene encoding the S. pombe glutathione transporter (Pgt1) by a genetic strategy that exploited the requirement of the cys1a, strain of S. pombe (which is defective in cysteine biosynthesis) for either cysteine or glutathione, for growth. Disruption of the ORF in the cys1a, strain led to an inability to grow on glutathione as a source of cysteine. Cloning and subsequent biochemical characterization of the ORF revealed that a high-affinity transporter for glutathione (Km=63 ,M) that was found to be localized to the plasma membrane. The transporter was specific for glutathione, as significant inhibition in glutathione uptake could be observed only by either reduced or oxidized glutathione, or glutathione conjugates, but not by dipeptides or tripeptides. Furthermore, although glu,cys,gly, an analogue of glutathione (,-glu,cys,gly), could be utilized as a sulphur source, the growth was not Pgt1 dependent. This further underlined the specificity of this transporter for glutathione. The strong repression of pgt1+ expression by cysteine suggested a role in scavenging glutathione from the extracellular environment for the maintenance of sulphur homeostasis in this yeast. [source] Localization of gene products using a chromosomally tagged GFP-fusion library in the fission yeast Schizosaccharomyces pombeGENES TO CELLS, Issue 2 2009Aki Hayashi We constructed a library of chromosomally-tagged green fluorescent protein (GFP) fusions in the fission yeast Schizosaccharomyces pombe. This library contains 1058 strains. In each strain, the coding sequence of GFP is integrated at the 3,-end of a particular chromosomal ORF such that the full-length GFP fusion construct is expressed under the control of the original promoter. Integration of the GFP coding sequence at the authentic chromosomal location of each gene was confirmed by PCR. Microscopic screening of these strains detected sufficient levels of GFP signal in 710 strains and allowed assignment of these GFP-fusion gene products with their intracellular localization: 374 proteins were localized in the nucleus, 65 proteins in the nucleolus, 34 proteins at the nuclear periphery, 27 proteins at the plasma membrane and cytoplasmic membranous structures, 24 proteins at the spindle pole body and microtubules, 92 proteins at cytoplasmic structures, and 94 proteins were uniformly distributed throughout the cytoplasm. [source] Rho1-GEFs Rgf1 and Rgf2 are involved in formation of cell wall and septum, while Rgf3 is involved in cytokinesis in fission yeastGENES TO CELLS, Issue 12 2005Tadashi Mutoh The Rho GTPase acts as a binary molecular switch by converting between a GDP-bound inactive and a GTP-bound active conformational state. The guanine nucleotide exchange factors (GEFs) are critical activators of Rho. Rho1 has been shown to regulate actin cytoskeleton and cell wall synthesis in the fission yeast Schizosaccharomyces pombe. Here we studied function of fission yeast RhoGEFs, Rgf1, Rgf2, and Rgf3. It was shown that these proteins have similar molecular structures, and function as GEFs for Rho1. Disruption of either rgf1 or rgf2 did not show a serious effect on the cell. On the other hand, disruption of rgf3 caused severe defects in contractile ring formation, F-actin patch localization, and septation during cytokinesis. Rgf1 and Rgf2 were localized to the cell ends during interphase and the septum. Rgf3 formed a ring at the division site, which was located outside the contractile ring and inside the septum where Rho1 was accumulated. In summary, Rgf1 and Rgf2 show functional redundancy, and roles of these RhoGEFs are likely to be different from that of Rgf3. Rho1 is likely to be activated by Rgf3 at the division site, and involved in contractile ring formation and/or maintenance and septation. [source] Meiosis induced by inactivation of Pat1 kinase proceeds with aberrant nuclear positioning of centromeres in the fission yeast Schizosaccharomyces pombeGENES TO CELLS, Issue 8 2004Yuji Chikashige Nuclear organization of chromosomes proceeds with significant changes during meiosis. In the fission yeast Schizosaccharomyces pombe, centromeres are clustered at the spindle-pole body (SPB) during the mitotic cell cycle; however, during meiotic prophase telomeres become clustered to the SPB and centromeres dissociate from the SPB. We followed the movement of telomeres, centromeres and sister chromatids in living S. pombe cells that were induced to meiosis by inactivation of Pat1 kinase (a key negative regulator of meiosis). Time-course observation in living cells determined the temporal order of DNA synthesis, telomere clustering, centromere separation and meiotic chromosome segregation. When meiosis was induced by Pat1 inactivation at the G1 phase of mitosis, telomeres clustered to the SPB as per normal meiosis, but in most cells the centromeres remained partially associated with the SPB. When meiosis was initiated at the G2 phase by Pat1 inactivation, both telomeres and centromeres retained their mitotic nuclear positions in the majority of cells. These results indicate that the progression of meiosis induced by Pat1 inactivation is aberrant from normal meiosis in some events. As Pat1 inactivation is often useful to induce S. pombe cells synchronously into meiosis, the temporal order of chromosomal events determined here will provide landmarks for the progression of meiosis downstream the Pat1 inactivation. [source] Calcineurin phosphatase in signal transduction: lessons from fission yeastGENES TO CELLS, Issue 7 2002Reiko Sugiura Calcineurin (protein phosphatase 2B), the only serine/threonine phosphatase under the control of Ca2+/calmodulin, is an important mediator in signal transmission, connecting the Ca2+ -dependent signalling to a wide variety of cellular responses. Furthermore, calcineurin is specifically inhibited by the immunosuppressant drugs cyclosporin A and tacrolimus (FK506), and these drugs have been a powerful tool for identifying many of the roles of calcineurin. Calcineurin is enriched in the neural tissues, and also distributes broadly in other tissues. The structure of the protein is highly conserved from yeast to man. The combined use of powerful genetics and of specific calcineurin inhibitors in fission yeast Schizosaccharomyces pombe (S. pombe) identified new components of the calcineurin pathway, and defined new roles of calcineurin in the regulation of the many cellular processes. Recent data has revealed functional interactions in which calcineurin phosphatase is involved, such as the cross-talk between the Pmk1 MAP kinase signalling, or the PI signalling. Calcineurin also participates in membrane traffic and cytokinesis of fission yeast through its functional connection with members of the small GTPase Rab/Ypt family, and Type II myosin, respectively. These findings highlight the potential of fission yeast genetic studies to elucidate conserved elements of signal transduction cascades. [source] Phosphatidylinositol 3-phosphate 5-kinase is required for the cellular response to nutritional starvation and mating pheromone signals in Schizosaccharomyces pombeGENES TO CELLS, Issue 2 2002Masayo Morishita Background: Phosphatidylinositol (3,5) bisphosphate, which is converted from phosphatidylinositol 3-phosphate by phosphatidylinositol 3-phosphate 5-kinase, is implicated in vacuolar functions and the sorting of cell surface proteins within endosomes in the endocytic pathway of budding yeast. A homologous protein, SpFab1p, has been found in the fission yeast Schizosaccharomyces pombe, but its role is not known. Results: Here we report that SpFab1p is encoded by ste12+ known as a fertility gene in S. pombe. The ste12 mutant grew normally under stress-free conditions, but was highly vacuolated and swelled at high temperatures and under starvation conditions. In nitrogen-free medium, ste12 cells were arrested in G1 phase, but partially defective in the expression of genes responsible for mating and meiosis. The ste12 mutant was defective both in the production of, and in the response to, mating pheromones. The amount of the pheromone receptor protein Map3p, was substantially decreased in ste12 cells. Map3p was transported to the cell surface, then internalized and eventually transported to the vacuolar lumen, even in the ste12 mutant. Conclusion: The results indicate that phosphatidylinositol(3,5)bisphosphate is essential for cellular responses to various stresses and for the mating pheromone signalling under starvation conditions. [source] Characterization of GTPase-activating proteins for the function of the Rho-family small GTPases in the fission yeast Schizosaccharomyces pombeGENES TO CELLS, Issue 12 2001Kentaro Nakano Background The small GTPase Rho1 has been shown to regulate the organization of the actin cytoskeleton and formation of the cell wall in the fission yeast Schizosaccharomyces pombe. Activity of Rho1 must be precisely regulated in vivo, since both increases and decreases in its activity affect cell growth and shape. Thus, it is important to clarify the mechanism by which the activity of Rho1 is regulated in vivo. Results Seven genes encoding putative GAPs, GTPase-activating proteins, for the function of the Rho-family proteins were isolated from S. pombe. After disruption of these genes, rga1+ was found to play important roles in cell growth and morphogenesis. In rga1 null cells, delocalized F-actin patches and extraordinary thickening of the cell wall and the septum were observed. On the other hand, over-expression of Rga1 produced shrunken or dumpy cells. The phenotype of the rga1 null cells or the Rga1-over-expressing cells was similar to that of cells containing abnormally high or low Rho1 activity, respectively. Moreover, direct association of Rga1 with Rho1 was shown. Rga1 was localized to the cell ends and septum where Rho1 is known to function. Conclusions In S. pombe, Rga1 is involved in the F-actin patch localization, cell morphogenesis, regulation of septation, and cell wall synthesis, probably functioning as a GAP for the function of Rho1. [source] Shotgun proteomic analysis of the microsomal fraction of eukaryotic cells using a two-dimensional reversed-phase×ion-pair reversed-phase HPLC setupJOURNAL OF SEPARATION SCIENCE, JSS, Issue 8 2009Martin Wörner Abstract A RP×IP-RP HPLC separation scheme was combined with on-line ESI-IT tandem MS or off-line MALDI tandem TOF MS and applied to the analysis of eukaryotic subcellular proteomes. Previous proteomic studies [1] were complemented by the approval of the approach to eukaryotic proteomes using the fission yeast Schizosaccharomyces pombe. The major focus was set to the analysis of primary human hepatocyte microsomes, representing a compartment of high interest due to its involvement in xenobiotic detoxification and cholesterol homeostasis. Of the 588 proteins identified from two donors, 24% are involved in cholesterol homeostasis or xenobiotic/lipid metabolism. Up to 50% of the identified proteins belong to the group of membrane proteins, difficult to investigate using gel-based proteomic approaches. We further demonstrated the reproducibility and comparability of the approach and reduced the amount of sample load by almost 70% with only minor loss of information about the proteins identified in the samples. The presented study clearly demonstrates the good applicability of the experimental setup to the analysis of subcellular proteomes including large membrane fractions, where only low amounts of sample material are available. [source] Kin1 is a plasma membrane-associated kinase that regulates the cell surface in fission yeastMOLECULAR MICROBIOLOGY, Issue 5 2010Angela Cadou Summary Cell morphogenesis is a complex process that depends on cytoskeleton and membrane organization, intracellular signalling and vesicular trafficking. The rod shape of the fission yeast Schizosaccharomyces pombe and the availability of powerful genetic tools make this species an excellent model to study cell morphology. Here we have investigated the function of the conserved Kin1 kinase. Kin1-GFP associates dynamically with the plasma membrane at sites of active cell surface remodelling and is present in the membrane fraction. Kin1, null cells show severe defects in cell wall structure and are unable to maintain a rod shape. To explore Kin1 primary function, we constructed an ATP analogue-sensitive allele kin1-as1. Kin1 inhibition primarily promotes delocalization of plasma membrane-associated markers of actively growing cell surface regions. Kin1 itself is depolarized and its mobility is strongly reduced. Subsequently, amorphous cell wall material accumulates at the cell surface, a phenotype that is dependent on vesicular trafficking, and the cell wall integrity mitogen-activated protein kinase pathway is activated. Deletion of cell wall integrity mitogen-activated protein kinase components reduces kin1, hypersensitivity to stresses such as those induced by Calcofluor white and SDS. We propose that Kin1 is required for a tight link between the plasma membrane and the cell wall. [source] | |||||||||||||